Non-canonical functions of the tuberous sclerosis complex-Rheb signalling axis

The protein products of the tuberous sclerosis complex (TSC) genes, TSC1 and TSC2, form a complex, which inhibits the small G-protein, Ras homolog enriched in brain (Rheb). The vast majority of research regarding these proteins has focused on mammalian Target of Rapamycin (mTOR), a target of Rheb. Here, we propose that there are clinically relevant functions and targets of TSC1, TSC2 and Rheb, which are independent of mTOR. We present evidence that such non-canonical functions of the TSC-Rheb signalling network exist, propose a standard of evidence for these non-canonical functions, and discuss their potential clinical and therapeutic implications for patients with TSC and lymphangioleiomyomatosis (LAM)

Rabin8 Protein Interacts with GTPase Rheb and Inhibits Phosphorylation of Ser235/Ser236 in Small Ribosomal Subunit Protein S6

The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that in association with Raptor, mLST8, PRAS40 and Deptor forms a complex (mTORC1) playing the key role in the regulation of protein biosynthesis, transcription, cellular metabolism, apoptosis and autophagy; mainly via direct phosphorylation of S6 kinases. mTORC1 is activated by growth factors and amino acids via the activation of Rheb GTPase. In the current study, we demonstrate for the first time that the over-expression of Rabin8, which functions as a guanine nucleotide exchange factor for Rab8 GTPase, suppresses phosphorylation of Ser235/Ser236 in ribosomal protein S6. Downregulation of Rabin8 using small interfering RNA (siRNA) increases the phosphorylation of Ser235/Ser236 in ribosomal protein S6. Furthermore, Rabin8 can be immunoprecipitated with Rheb GTPase. These results suggest the existence of a novel mechanism of mTORС1 regulation and its downstream processes. Since Rabin8 is a known regulator of ciliogenesis, a potential link can exist between regulation of Rheb/mTORC1 and ciliogenesis

Engineering Regulation in Anaerobic Gut Fungi during Lignocellulose Breakdown

The development of a renewable, bio-based economy requires efficient methods to extract fermentable sugars from complex plant material. Currently, bioprocessing from crude biomass requires multiple steps including pretreatment to separate lignin from sugar-rich cellulose and hemicellulose, enzymatic hydrolysis to release simple sugars, and microbial fermentation to produce value-added chemicals. Consolidated bioprocessing seeks to improve bioprocessing efficiency by reducing the number of steps required to get from plant biomass to chemical product. To address this challenge, we derived inspiration from natural microbial communities known for degrading biomass. Within the rumen microbiome of large herbivores, anaerobic gut fungi are the primary colonizers of plant material and present an untapped opportunity for consolidated bioprocessing. These unique microorganisms efficiently hydrolyze lignocellulosic biomass into simple sugars, but remain relatively uncharacterized in comparison to industrial production organisms. We implemented Next Generation Sequencing (NGS) technologies alongside biochemical studies to develop a deeper understanding of gut fungi, their metabolism, and the mechanisms by which they break down complex biomass to identify a path forward for their industrial application. We also developed simple, rapid methodologies for cryopreservation and DNA extraction that are critical for the development of industrial microbes.Sequencing and functional annotation of transcriptomes and genomes of novel isolated species of gut fungi has elucidated their large repertoire of biomass degrading enzymes including cellulases, hemicellulases, and accessory enzymes. These enzymes allow them to efficiently degrade crude biomass, yielding similar growth rates on complex plant material and simple sugars. Remarkably, in isolated batch culture, the biomass degrading power of gut fungi is sufficient to generate surplus fermentable sugars for the growth of additional microorganisms. This ability has been exploited to develop a novel two-stage consolidated bioprocessing scheme that uses anaerobic gut fungi to consolidate the pretreatment and hydrolysis steps in traditional bioprocessing to hydrolyze sugars directly from crude biomass. These sugars can then be fed to the easily metabolically engineered model yeast, Saccharomyces cerevisiae, to support growth and bioproduction in a two-stage fermentation scheme. Further, RNA sequencing studies have provided critical insight into the regulation of biomass degrading activity. Gene expression during growth on varying substrates and in response to a carbon catabolite repressor has revealed conditions required to optimize expression of biomass degrading enzymes. Unannotated sequences that co-regulate with predicted biomass degrading enzymes have also been identified as candidate genes that may host novel biomass degrading function. Together these results reveal important process considerations for the use of gut fungi in industrial bioprocessing to maximize the production of enzymes and the degradation of biomass. While challenges remain for the implementation of gut fungi in industrial bioprocessing, we have demonstrated their potential to consolidate pretreatment and hydrolysis either through engineered culturing schemes or development of improved enzyme cocktails for biomass hydrolysis

The Genetics of Pneumothorax.

A genetic influence on spontaneous pneumothoraces-those occurring without a traumatic or iatrogenic cause-is supported by several lines of evidence: 1) pneumothorax can cluster in families (i.e., familial spontaneous pneumothorax), 2) mutations in the FLCN gene have been found in both familial and sporadic cases, and 3) pneumothorax is a known complication of several genetic syndromes. Herein, we review known genetic contributions to both sporadic and familial pneumothorax. We summarize the pneumothorax-associated genetic syndromes, including Birt-Hogg-Dubé syndrome, Marfan syndrome, vascular (type IV) Ehlers-Danlos syndrome, alpha-1 antitrypsin deficiency, tuberous sclerosis complex/lymphangioleiomyomatosis, Loeys-Dietz syndrome, cystic fibrosis, homocystinuria, and cutis laxa, among others. At times, pneumothorax is their herald manifestation. These syndromes have serious potential extrapulmonary complications (e.g., malignant renal tumors in Birt-Hogg-Dubé syndrome), and surveillance and/or treatment is available for most disorders; thus, establishing a diagnosis is critical. To facilitate this, we provide an algorithm to guide the clinician in discerning which cases of spontaneous pneumothorax may have a genetic or familial contribution, which cases warrant genetic testing, and which cases should prompt an evaluation by a geneticist

Loss of the Birt–Hogg–Dubé tumor suppressor results in apoptotic resistance due to aberrant TGFβ-mediated transcription

Birt–Hogg–Dubé (BHD) syndrome is an inherited cancer susceptibility disease characterized by skin and kidney tumors, as well as cystic lung disease, which results from loss-of-function mutations in the BHD gene. BHD is also inactivated in a significant fraction of patients with sporadic renal cancers and idiopathic cystic lung disease, and little is known about its mode of action. To investigate the molecular and cellular basis of BHD tumor suppressor activity, we generated mutant Bhd mice and embryonic stem cell lines. BHD-deficient cells exhibited defects in cell-intrinsic apoptosis that correlated with reduced expression of the BH3-only protein Bim, which was similarly observed in all human and murine BHD-related tumors examined. We further demonstrate that Bim deficiency in Bhd−/− cells is not a consequence of elevated mTOR or ERK activity, but results instead from reduced Bim transcription associated with a general loss of TGFβ-mediated transcription and chromatin modifications. In aggregate, this work identifies a specific tumor suppressive mechanism for BHD in regulating TGFβ-dependent transcription and apoptosis, which has implications for the development of targeted therapies

Constitutive mTOR activation in TSC mutants sensitizes cells to energy starvation and genomic damage via p53

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/102117/1/emboj7601900.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/102117/2/emboj7601900-sup-0001.pd

MicroRNA-21 is Induced by Rapamycin in a Model of Tuberous Sclerosis (TSC) and Lymphangioleiomyomatosis (LAM)

Lymphangioleiomyomatosis (LAM), a multisystem disease of women, is manifest by the proliferation of smooth muscle-like cells in the lung resulting in cystic lung destruction. Women with LAM can also develop renal angiomyolipomas. LAM is caused by mutations in the tuberous sclerosis complex genes (TSC1 or TSC2), resulting in hyperactive mammalian Target of Rapamycin (mTOR) signaling. The mTOR inhibitor, Rapamycin, stabilizes lung function in LAM and decreases the volume of renal angiomyolipomas, but lung function declines and angiomyolipomas regrow when treatment is discontinued, suggesting that factors induced by mTORC1 inhibition may promote the survival of TSC2-deficient cells. Whether microRNA (miRNA, miR) signaling is involved in the response of LAM to mTORC1 inhibition is unknown. We identified Rapamycin-dependent miRNA in LAM patient angiomyolipoma-derived cells using two separate screens. First, we assayed 132 miRNA of known significance to tumor biology. Using a cut-off of >1.5-fold change, 48 microRNA were Rapamycin-induced, while 4 miRs were downregulated. In a second screen encompassing 946 miRNA, 18 miRs were upregulated by Rapamycin, while eight were downregulated. Dysregulation of miRs 29b, 21, 24, 221, 106a and 199a were common to both platforms and were classified as candidate “RapamiRs.” Validation by qRT-PCR confirmed that these microRNA were increased. miR-21, a pro-survival miR, was the most significantly increased by mTOR-inhibition (p<0.01). The regulation of miR-21 by Rapamycin is cell type independent. mTOR inhibition promotes the processing of the miR-21 transcript (pri-miR-21) to a premature form (pre-miR-21). In conclusion, our findings demonstrate that Rapamycin upregulates multiple miRs, including pro-survival miRs, in TSC2-deficient patient-derived cells. The induction of miRs may contribute to the response of LAM and TSC patients to Rapamycin therapy